The relief of chronic itch (pruritus) would improve the quality of life of tens of millions of people in the US. Pruritus is poorly controlled by antihistamines but histamine-independent receptors have only recently been identified as potential targets for anti-pruritics. These newly identified pruritic receptors include the Mas-related G protein receptors, Toll-like receptors, and the thymic stromal lymphopoietin receptor, and have been found in different subsets of rodent sensory neurons. Presently, the localization and signaling mechanisms of homologous itch receptors in human sensory neurons are unknown, creating potential obstacles for translation. For this proposal we have developed a novel strategy to establish target validity at a preclinical stage by performing physiological studies in viable human sensory neurons in vitro. The major objectives of this proposal are to 1) understand the transduction of histaminergic and non-histaminergic itch stimuli in characterized human sensory neurons, and 2) to determine whether keratinocytes obtained from donors with atopic dermatitis control sensitization of pruriceptive neurons. Human sensory neurons will be characterized based on functional responses to pruritogens and on gene expression profile. Pruritic receptors have been shown to couple functionally and selectively to Transient Receptor Potential (TRP) channels in rodents. We will test whether human TRP channels are likewise gated by pruritic receptor activation in human sensory neurons and will examine whether other classes of ion channels couple to human pruritic receptors. In Aim 2 our goal is to understand sensitization of pruriceptive sensory neurons, a phenomenon that can explain the ongoing and heightened itch experienced by individuals with chronic pruritic conditions including atopic dermatitis. We hypothesize that atopic human keratinocytes will control pruritic receptor function and excitability of human sensory neurons. For this aim we develop novel human sensory neurons and human keratinocytes co-culture techniques. Our proposal represents the first steps toward a broader implementation of in vitro human sensory physiology. This strategy will provide valuable preclinical assessment on the feasibility of targeting novel receptors in human for itch and pain, and reduce the risks involved in clinical trials.